Preclinical Evaluation of IMGC936, a Next-Generation Maytansinoid-based Antibody-drug Conjugate Targeting ADAM9-expressing Tumors.

ADAM metallopeptidase domain 9 (ADAM9) is a member of the ADAM family of multifunctional, multidomain type 1 transmembrane proteins. ADAM9 is overexpressed in many cancers, including non-small cell lung, pancreatic, gastric, breast, ovarian, and colorectal cancer, but exhibits limited expression in normal tissues. A target-unbiased discovery platform based on intact tumor and progenitor cell immunizations, followed by an IHC screen, led to the identification of anti-ADAM9 antibodies with selective tumor-versus-normal tissue binding.

Preclinical Development of MGC018, a Duocarmycin-based Antibody-drug Conjugate Targeting B7-H3 for Solid Cancer.

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival.

Multispecific, Multivalent Antibody-Based Molecules Engineered on the DART® and TRIDENT Platforms.

Multispecific antibodies bind two or more different antigens and enable new therapeutic applications that cannot be replicated with conventional monoclonal antibodies, such as bridging different cells or bringing soluble proteins in close proximity. The DART and TRIDENT platforms enable the engineering of such antibodies. A DART molecule combines two independent antigen-binding sites in a stabilized, diabody-like structure.

Development of MGD007, a gpA33 x CD3-Bispecific DART Protein for T-Cell Immunotherapy of Metastatic Colorectal Cancer.

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells.

A CD3xCD123 bispecific DART for redirecting host T cells to myelogenous leukemia: preclinical activity and safety in nonhuman primates.

Current therapies for acute myeloid leukemia (AML) are largely ineffective, and AML patients may benefit from targeted immunotherapy approaches. MGD006 is a bispecific CD3xCD123 dual-affinity re-targeting (DART) molecule that binds T lymphocytes and cells expressing CD123, an antigen up-regulated in several hematological malignancies including AML. MGD006 mediates blast killing in AML samples, together with concomitant activation and expansion of residual T cells.

Development of an Fc-enhanced anti-B7-H3 monoclonal antibody with potent antitumor activity.

Purpose: The goal of this research was to harness a monoclonal antibody (mAb) discovery platform to identify cell-surface antigens highly expressed on cancer and develop, through Fc optimization, potent mAb therapies toward these tumor-specific antigens.

Experimental Design: Fifty independent mAbs targeting the cell-surface immunoregulatory B7-H3 protein were obtained through independent intact cell-based immunizations using human tissue progenitor cells, cancer cell lines, or cell lines displaying cancer stem cell properties. Binding studies revealed this natively reactive B7-H3 mAb panel to bind a range of independent B7-H3 epitopes.

Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcγ receptor binding properties.

Introduction: Response to trastuzumab in metastatic breast cancer correlates with expression of the high binding variant (158V) of the activating Fcγ receptor IIIA (CD16A). We engineered MGAH22, a chimeric anti-HER2 monoclonal antibody with specificity and affinity similar to trastuzumab, with an Fc domain engineered for increased binding to both alleles of human CD16A.

Methods: MGAH22 was compared to an identical anti-HER2 mAb except for a wild type Fc domain.

Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma.

We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell-specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations.

Fibroblasts from PS1 mutated pre-symptomatic subjects and Alzheimer's disease patients share a unique protein levels profile.

In Alzheimer's disease (AD), a major goal is to improve early detection, as the diagnosis cannot be made until patients exhibit a noticeable decline in cognition and the brain is irreversibly damaged. With this aim in mind, we performed proteome analysis of familial AD fibroblasts from both demented and pre-symptomatic subjects, using a 2D-PAGE based approach and then identifying proteins by mass spectrometry. We compared primary fibroblast cultures from skin biopsy of presenilin 1 (PS1) mutated patients, pre-symptomatic subjects carrying mutations in the PS1 gene but healthy at the time of skin biopsy, and age-matched individuals as control.

Therapeutic control of B cell activation via recruitment of Fcgamma receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold.

Objective: To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications.

Methods: DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells.

Effector cell recruitment with novel Fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo B-cell depletion.

Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART).

New perspectives for the diagnosis of Alzheimer's disease.

Alzheimer's disease is the most common cause of dementia in the elderly. Currently its clinical assessment is based on the exclusion of other forms of dementia and a definitive diagnosis requires a confirmation by examination of post-mortem brains. Therefore, there is a strong need to find easy measurable AD biomarkers that could facilitate the early diagnosis and monitoring the efficacy of the few therapies currently available.

Occurrence of an anomalous endocytic compartment in fibroblasts from Sandhoff disease patients.

Sandhoff disease (SD) is a lysosomal storage disorder due to mutations in the gene encoding for the beta-subunit of beta-hexosaminidase, that result in beta-hexosaminidase A (alphabeta) and beta-hexosaminidase B (betabeta) deficiency. This leads to the storage of GM2 ganglioside in endosomes and lysosomes, which ends in a progressive neurodegeneration. Currently, very little is known about the biochemical pathways leading from GM2 ganglioside accumulation to pathogenesis.

Identification and characterization of mature beta-hexosaminidases associated with human placenta lysosomal membrane.

Hex (beta-hexosaminidase) is a soluble glycohydrolase involved in glycoconjugate degradation in lysosomes, however its localization has also been described in the cytosol and PM (plasma membrane). We previously demonstrated that Hex associated with human fibroblast PM as the mature form, which is functionally active towards G(M2) ganglioside. In the present study, Hex was analysed in a lysosomal membrane-enriched fraction obtained by purification from highly purified human placenta lysosomes.

Transfection of cultured eukaryotic cells using cationic lipid reagents.

The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipid-based transfection reagents has accelerated biology research in studies of gene expression, control of cell growth, and cell lineage. In this unit, DNA transfection is described for adherent mammalian cells (both cells and primary cultures) along with an alternate procedure for enhanced transfection. A protocol is also described for transfection of suspension cells (lymphoid, myeloid and leukemic-derived cells).

Transfection of cultured eukaryotic cells using cationic lipid reagents.

The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. This unit describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using cationic lipid reagents.

Transfection of cultured eukaryotic cells using cationic lipid reagents.

The development of high-efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. Replacing our previous unit on this topic, this new version describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using the cationic lipid system.

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells.

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter.

Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications.

Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposome-DNA complexing time, and the presence or absence of media components such as antibiotics and serum. The importance of these factors in Lipofectamine 2000 mediated transfection will be discussed together with some specific applications: transfection of primary neurons, high throughput transfection, and delivery of small interfering RNAs.

Improving the transfection efficiency of post-mitotic neurons.

The transfection of post-mitotic cells, including primary cortical and hippocampal neurons, has proven for the most part to be inefficient. Methods such as DNA/Ca++ phosphate co-precipitation, electroporation, cationic lipids and micro-injection are often toxic to the cell and rarely give a transfection efficiency exceeding 3% of the surviving culture. Virus transfection methods using modified viruses such as adeno and semliki-forest virus have been shown to be more efficient but the procedures are often time consuming and virus infections may interfere with protein processing.

The SFV Gene Expression System.

The SFV Expression System is a DNA expression system used to produce recombinant protein in eukaryotic cells (1,2). The SFV system is based on the Semliki Forest Vnus (SFV), which has several features that provide distinct advantages for a good cDNA expression system. These are: 1.

Generation of Recombinant Baculovirus DNA in E.coli Using a Baculovirus Shuttle Vector.

Baculovirus vectors are now widely used to direct the expression of heterologous genes in cultured insect cells and insect larvae. In most cases, heterologous genes placed under transcriptional control of the polyhedrm promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) are abundantly expressed during the late stages of infection. The recombinant proteins are usually soluble and functionally similar to their authentic counterparts (l-7).

Febrile and afebrile convulsions: a clinical follow-up.

A joint study was performed on 204 children who suffered separate febrile (FS) and afebrile seizures (aFS) within a short space of time to evaluate the risk of subsequent convulsive crises. The data obtained revealed frequent recurrence of seizures and high risk of subsequent convulsive afebrile crises in these children.

Identification of enhancer-like elements in human IFN-gamma genomic DNA.

We have previously shown that transfection of a plasmid clone containing full length human IFN-gamma genomic DNA into a murine T-lymphoblastoid line is followed by basal expression of the transfected gene, with increased transcription occurring upon stimulation of the cells with either phorbol ester or IL-2. In addition, upon transfection of this DNA into murine fibroblasts, high level constitutive transcription was observed. In contrast to the results obtained under tissue culture conditions, introduction of the same DNA into the mouse germline resulted in tissue-specific expression of the transgene.

Expression of human IFN-gamma genomic DNA in transgenic mice.

We have introduced an 8.6-kb fragment of human genomic DNA containing the full length IFN-gamma gene into the mouse germline. The transgenic animals had no biologic or developmental defects as human IFN-gamma does not bind to the mouse IFN R.